Field of the Invention
The embodiments disclosed herein relate to molecular diagnostics, and, in particular, diagnostics used to detect and identify microbes carrying extended spectrum beta lactamases (ESBLs), and in particular CTX-M genes.
Description of the Related Art
βlactamases confer resistance against β-lactam drugs. These enzymes destroy the β-lactam ring of the β-lactam antibiotics, such as penicillin, cephalosporins, cephamycins, and carbapenems (ertapenem). These antibiotics have a common element in their molecular structure: a four-atom ring known as a beta-lactam. The lactamase enzyme breaks that ring open, deactivating the molecule's antibacterial properties.
Extended spectrum β-lactamases (ESBLs) are increasingly responsible for nosocomial infections arising around the globe, and alarmingly, for community emergence as well. (Rossolini et al. 2008, CMI). ESBLs are beta-lactamases that hydrolyze extended-spectrum cephalosporins with an oxyimino side chain. These cephalosporins include cefotaxime, ceftriaxone, and ceftazidime, as well as the oxyimino-monobactam aztreonam. Thus ESBLs confer resistance to these antibiotics and related oxyimino-beta lactams. The most well-known ESBLs are derived from the TEM-1, TEM-2, or SHV-1 genes, and include mutations that alter the amino acid configuration around the active site of these β-lactamases. This extends the spectrum of β-lactam antibiotics susceptible to hydrolysis by these enzymes.
TEM and SHV Classical variants, such as TEM and SHV, are actually spreading rapidly across the United States of America after having affected most of Europe, while a new type of ESBLs, CTX-M, is prevalent in South America, Mediterranean and Eastern European countries (Govinden et al. 2007, AJB). The latest, which owns its name to its high activity against cefotaxime, was observed in the late 1980s in Japan, Europe and Argentina, most specifically in Germany in 1989 (Naas et al. 2008, CMI). It is considered to be the most successful group of all (Rasmussen & Hoiby 2004, CJM). Its appearance could be a consequence of the increased use of ceftriaxone and/or cefotaxime to treat bacterial infections, and its origin is known to be from chromosomal genes resident in members of the genus Kluyvera. To this day, over 85 CTX-M derivatives, classified in 5 phylogenetic groups (CTX-M-1, 2, 8, 9 and 25), have been documented according to the Lahey Clinic website, accessible at the world-wide web address lahey.org/Studies.
CTX-M resistance genes are found in Enterobacteriaceae and can be transmitted through plasmids between species easily. Enterobacterial species including Klebsiella pneumoniae, Escherichia coli, and the like possessing the CTX-M genes are considered to be the main cause for urinary tract infection. Other Enterobacteriaceae, such as Enterobacter cloacae, Proteus mirabilis, Salmonella enterica, Enterobacter aerogenes, as well as Klebsiella oxytoca, can also harbor CTX-M genes. Detection of CTX-M resistant strains is especially crucial, as it requires isolation from other patients in hospitals, and would leave only carbapenems as the main treatment for infections.
Until recently, the only way to know a strain's resistance was to perform a manual antimicrobial susceptibility testing. Susceptibility tests suffer from many drawbacks, including the amount of time to obtain a result, i.e., between 48 to 96 hours. First, the operator needs to isolate the bacterial strain from the specimen, which could take up to 48 hours; then proceed with the biochemical identification, which is another 18 to 24 hours, and then with the manual antimicrobial susceptibility testing, which could also take up to 24 hours. In addition to the delay in obtaining results, manual testing methods also suffer from other problems, such as lack of reproducibility due to improper storage of antibiotic disks, improper diffusion of some antibiotic disks, and a lack into the standardization of the process.
The specificity and accuracy of ESBLs detection is critical, as false negative results can lead medical practitioners to design an inappropriate antibiotic regimen, e.g., treatment of an individual with an ESBL infection with third-generation cephalosporins or with aztreonam. This is poses unnecessary risks to the treated individual, and also increases the odds of cross-contamination within a clinical setting, e.g., a hospital. As some strains producing ESBLs will not show in vitro resistance to all third- or fourth-generation cephalosporins using the suggested breakpoints, the Clinical and Laboratory Standard Institute, (CLSI), recommends reporting ESBL-producing Enterobacteriaceae as resistant to penicillins, cephalosporins and aztreonam, because they might end up being clinically resistant (CLSI, M100-S18). The ability of organisms that harbor CTX-M resistance genes to hydrolyze the newer cephalosporins and aztreonam renders their detection even more difficult. CLSI guidelines pose the threat of misdiagnosing the presence of CTX-M-producing strains, depending on the drugs used in both the initial screening and confirmation tests.
The embodiments disclosed herein provide advantages over other methods used to detect and identify bacteria that have ESBLs, e.g., CTX-M resistance genes, including improved specificity, availability of results in a shorter time period, and eliminates the need to perform additional steps, such as agarose gel electrophoresis, to detect ESBLs. (Lartigue et al. 2004, FEMS ML; Pitout et al. 2004, JCM; Pitout et al. 2007, CMI). Furthermore, the embodiments disclosed herein offer advantages over other reported methods for the detection of ESBLs, including CTX-M, in that methods and compositions disclosed herein are specifically designed for the detection and identification newly discovered isoforms of the CTX-M gene, which were not known as of the time of the development of assays described, for example in U.S. Patent Application Publication No. US20070248954. The methods disclosed in US20070248954 use primers that are not fully complementary to the sequences of the newly discovered CTX-M isoforms, which could compromise specificity, or even result in false negative results.